1a.Objectives (from AD-416):
1) The identification of HST-host gene interactions using purified toxins and wheat mapping populations;
2) Identification of proteinaceous toxin genes using purified toxins in conjunction with mass spec analysis to identify candidate genes for further evaluation;
3) Verification of candidate genes using heterologous expression, transformation, and site directed gene disruption; and
4) Mode of actions studies to identify the molecular and biochemical mechanism whereby the toxin is instrumental in causing disease, including protein-protein interaction studies, inhibitor studies, and protein localization studies.
1b.Approach (from AD-416):
Stagonospora nodorum will be used to produce extracts of the proteinaceous host-selective toxins (HSTs) that we have identified. HSTs will be purified and active fractions will be used to identify protein sequences using mass-spectrometry. Each of the presently characterized toxins has been shown to be a protein that interacts with a corresponding host sensitivity gene in an inverse gene-for-gene manner. Candidate genes will be revealed using the S. nodorum genome sequence. Additionally, bioinformatics will be used to scan the S. nodorum genome to identify candidate genes based on signal sequence domains and predicted protein size in addition to other relevant criteria. We have isolated a nonpathogenic strain of S. nodorum that appears to secrete no toxins. We will use this strain to verify toxin gene candidates. Candidate genes will initially be expressed in this isolate to verify toxin production and pathogenicity changes. Lastly, we will continue to identify new toxins using an international S. nodorum collection. This proposal will enhance economic opportunities for agricultural producers. This will be accomplished by providing valuable information to scientists including breeders for the improvement of wheat as a food crop especially as it relates to providing durable resistance sources to growers.
Characterization of SnTox1 mode of action and localization – SnTox1 has significant homology to other chitin binding proteins, and we have shown that SnTox1 physically binds chitin as well other polysaccharides found in the plant cell wall. These polysaccharides include cellulose, xylan, chitosan, crab shell chitin, and shrimp shell chitin. Using an SnTox1-GFP fusion, we showed that SnTox1 localized to the outside of the fungal mycelium as well as to the plant cell wall. Using a His tag, SnTox1 was purified and treated with a fluorescent label for localization. Wheat plants harboring Snn1, the corresponding wheat sensitivity gene, were inoculated and cross sections were made in order to visualize where SnTox1 was localized to during the infection process. Preliminary data indicate that SnTox1 is able to induce necrosis in the mesophyll cells, while remaining localized to the top of the epidermal layer of the plant.
Necrotrophic effector candidate gene identification – Continued work on a necrotrophic effector candidate gene list has been useful in identifying several new NE candidates that induce necrosis on certain susceptible cultivars in a host specific manner. These genes have been expressed in Pichia pastoris (yeast) and have been shown to induce necrosis. Further work in validating these genes is in progress.